JP2002286639A - Time-resolved fluorescence detecting device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a time-resolved fluorescence detecting device capable of acquiring delayed fluorescence images without scattering noise. SOLUTION: The time-resolved fluorescence detecting device is provided with an exciting light intermittent emergence part for intermittently shining a flux of exciting light with predetermined timing, an optical path for the flux of exciting light shone from the exciting light intermittent emergence part, a phase resolving part provided on the optical path for resolving the phase of the flux of exciting light, an optical system for acquiring fluorescence generated by excitation by the flux of exciting light, and a control part arranged in such a way as to connect to both the exciting light intermittent emergence part and the optical system for controlling the emergence timing and the irradiation time of the flux of exciting light by the exciting light intermittent emergence part and the acquisition timing of the fluorescence by the optical system.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a time-resolved fluorescence measuring device. In particular, the present invention relates to a time-resolved fluorescence image measurement device suitable for use in analysis of a DNA chip or a DNA microarray.

[0002]

2. Description of the Related Art A time-resolved fluorescence analysis detector recently developed uses a delayed fluorescent substance as a labeling substance, and obtains a long-lived fluorescent light generated by irradiating it with excitation light with a certain time delay to obtain a sample. This is a technique for selectively measuring the fluorescence intensity of light. For example, JP-A-9-43146 and JP-A-5-188001 disclose measurement target light from a phosphor by separation optics to detect the delayed fluorescence, and also measure the intermittent light beam from the excitation light beam discontinuous emission unit. A technique for performing time-resolved measurement by irradiating a sample and a delay line with separation by a beam splitter is disclosed. Japanese Patent Application Laid-Open No. H10-267844 discloses a time-resolved fluorescence detector that includes an excitation light beam intermittent emission unit, a delayed fluorescence acquisition unit that allows only the fluorescence from the sample to pass, and a photoelectric conversion unit that measures the fluorescence intensity.

[0003]

SUMMARY OF THE INVENTION The present invention proposes to use the above-mentioned time-resolved fluorescence detection, particularly as a means for analyzing a DNA chip or a DNA microarray.

[0004] DNA chip or DNA to be detected
A microarray is a device capable of obtaining enormous information from a microdevice. As a means for analyzing a DNA chip and a DNA microarray, for example, a system using a laser fluorescence microscope has been disclosed (Nature, 346, 555-55).
6, 1993). However, the conventional analysis method including such a method has a problem that an extremely large artifact is generated, and also has a problem that the detection sensitivity and the detection accuracy associated with the detection of the fluorescent substance are insufficient. The inventors have focused on using time-resolved fluorescence detection as a means for solving these problems.

[0005] Time-resolved fluorescence detection is a means for selectively measuring the target fluorescence intensity by using a delayed fluorescent substance as a labeling substance, as described above. However, conventionally, there is no device that can be used for measuring a microsystem such as a DNA chip or a DNA microarray, and it is impossible to obtain sufficient sensitivity and accuracy using such a conventional device. is there. Accordingly, the present inventors have conducted research for the development of a highly sensitive and highly accurate time-resolved fluorescence detection apparatus that can be used especially for trace systems such as DNA chips and DNA microarrays.

As a result, the following problems have become apparent. In other words, it has been clarified that, when a laser is used as the light source of the intermittent excitation light emitting section in the conventional apparatus, speckle noise due to the coherence is generated, thereby generating scattering noise. Therefore, as described above, the present invention provides a time-resolved fluorescence detection method using a DNA chip and a DN.
Focusing on the use of A microarray, it is based on finding a problem in such use.

[0007] That is, an object of the present invention is to provide a time-resolved fluorescence measurement apparatus capable of acquiring a delayed fluorescence image free from scattering noise. In particular, oligo DNA probes or c
An object of the present invention is to provide a fluorescence detection means for a DNA chip or DNA microarray on which DNA is immobilized.

[0008]

Means for Solving the Problems In order to achieve the above object, the present inventor has found the following means as a result of earnest studies. That is, a time-resolved fluorescence detection device,
An excitation light intermittent emission unit that intermittently emits an excitation light beam at a predetermined timing, an optical path for the excitation light beam emitted from the excitation light intermittent emission unit, and a phase of the excitation light beam provided on the optical path. And an optical system for acquiring fluorescence generated by excitation by the excitation light beam, and the excitation light intermittent emission unit disposed and connected to both the excitation light intermittent emission unit and the optical system. A control unit for controlling the emission timing and irradiation time of the excitation light beam by the unit, and the acquisition timing of the fluorescence by the optical system,
It is a time-resolved fluorescence detection device comprising: Here, the optical system includes a photoelectric conversion unit that measures the fluorescence as a fluorescence intensity image and uses the measured fluorescence as an electric signal. What is particularly important is that the phase eliminating unit is an optical fiber bundle.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The apparatus of the present invention will be described below. Here, a preferred embodiment of the time-resolved fluorescence measuring apparatus of the present invention is shown as an apparatus for detecting fluorescence in a DNA chip or a DNA microarray. The device of the present invention is a DNA chip or DN which is a micro device.
Particularly useful for A microarrays, however, the targets of detection are not limited to these microdevices, but will also show significant effects on a wide range of other targets as well.

[Time-Resolved Fluorescence Measurement Apparatus] FIG. 1 shows a preferred example of the time-resolved fluorescence measurement apparatus of the present invention. The intermittent emission part of the excitation light of the present apparatus is preferably a light source capable of substantially continuous lighting, and in particular, the pulse laser 1 can be preferably used. The pulse laser 9 has a short pulse width, a small amount of jitter and a high output. For example, Nd:
A YAG / YLF laser, Nd: KGW laser, Nd: glass laser, nitrogen / dye laser, dye laser, tunable solid-state laser and the like are used.

The optical path of the apparatus preferably uses an optical fiber. A collimator lens unit 2 is connected to the pulse laser 1, and a light guide optical fiber 3 is coupled to the collimator lens unit 2 (FIG. 1). Further, the light guide optical fiber 3 is connected to a phase canceling unit 4 described later, and further connected to a light source switching unit via the light guide optical fiber 5 (FIG. 1).

Light guide optical fibers 3 and 5
May be a commonly used optical fiber, and its thickness may be 0.5 μm to 100 μm in diameter, and can be changed as desired. Further, the light guide optical fibers 3 and 5 may be the same optical fiber or different optical fibers.

The light guide optical fiber 5 is connected to a light source switching section 8 by a relay lens unit 6. The light source switching unit 8 is designed to be able to switch to either a mercury lamp or a xenon lamp 7.

The optical system of the apparatus includes an optical unit 10 described below and a CCD camera 9 attached to the optical unit 10 for detecting fluorescence.
(FIG. 1). The fluorescence from the sample 11 passes through the objective lens built in the optical unit 10 and passes through the CCD camera 9.
(FIG. 1). The CCD camera measures the fluorescence as a fluorescence intensity image, and the measured data is converted into an electric signal by a photoelectric conversion unit.

The control section of the present invention comprises a delay circuit 12, a timing generation section 13, and an operation / display section 14.
The control section controls the operation timing of image acquisition by the CCD camera 9 and the light emission timing and light emission time of the pulse laser 1. The preferred operation timing will be described later in detail. The pulse of the pulse laser 1 is
It is caused by substantial blinking and continuous lighting of the light source. The optical system acquires fluorescence obtained after a certain time delay from pulse irradiation. Such control of the operation timing is performed by the control unit.

The operation / display unit 14 includes an operation unit and a display unit, and can issue an instruction according to a program of a necessary operation timing, and can display an image such as a result. The delay circuit 12 delays the pulse output based on the delay amount specified by the operation unit, that is, the delay time. In accordance with the delay signal of the delay circuit 12 and the instruction from the calculation unit, the timing generation unit 13 is controlled so that the fluorescence obtained by delaying a predetermined time by the light beam passage timing of the excitation light interrupting unit is passed by a mechanical chopper. ing.

[Phase Elimination Unit] For the phase elimination unit, for example, an optical fiber bundle as shown in FIG. 3 can be used. FIG. 3 shows an example of each preferable dimension, and these numerical values are shown in units of mm.

The optical fiber bundle that can be used in the present invention can be constituted by combining a plurality of optical fibers 26 having different lengths into an aggregate. The optical fiber provided in the bundle part 26 is converged by the converging part 25 and connected to the light guide optical fiber of the present apparatus via the connecting part 24.

When the fiber in the bundle portion covers the ultraviolet region, it is preferable that the material of the fiber is quartz. However, the material is not limited to this, and may be made of any material generally used as an optical fiber. The length of each fiber is diversified by extending the length of the basic fiber plus 7 to 15 mm. The length of the basic fiber can be changed depending on the emission wavelength of the pulse laser used. By using an optical fiber bundle in which a plurality of fibers having different lengths are collected in this manner, it is possible to minimize speckle noise due to coherency. Thereby, fluorescence can be detected with high sensitivity and high accuracy.

The number of fibers included in the optical fiber bundle used in the apparatus of the present invention is about 1 to 50.
0 is preferable, 20 to 200 is practically preferable, and about 100 is particularly preferable. Bundle 2
The thickness of one optical fiber constituting 6 may be from 0.5 μm to 100 μm, and is preferably from 10 μm to 50 μm.

[Optical System] The CCD camera that can be used in the present apparatus may be any camera that can obtain a fluorescent image.

FIG. 2 shows an example of an optical system that can be used in the present invention. The light source switching mirror 17 normally emits a light beam in the state 1 at a position indicated by a solid line. When the light source switching mirror is set to the state 2 indicated by a broken line as desired, the sample is irradiated with the pulse laser light through the relay lens, reflected by the dichroic mirror 18, and passed through the objective lens 23. In this case, since the light source is monochromatic laser light before the dichroic mirror 18, a wavelength selecting filter is not required. Sample 1
The fluorescence generated from 1 passes through the dichroic mirror 18 again through the objective lens 23, further passes through the fluorescence selection filter 22, the imaging lens 21, and the intermediate zoom lens 20, and then forms an image as an image by the CCD camera. Is done.

The intermediate zoom lens can be determined by the illumination range of the sample 11 and the imaging range of the CCD camera.

Assuming that the number of visual fields is 22, the magnification mo of the objective lens is as follows: visual field range = number of visual fields / mo = 22/4 = 5.5 (mo = 4)
When the number of fields of view is 22, the intermediate magnification ratio mc is as follows: imaging range = number of fields × mc = 22 × 0.5 = 11 (mc =
0.5) and an image of 5.5 mm in diameter is formed as an area of 11 mm in diameter on the CCD camera. It is possible to determine the intermediate magnification ratio according to the size of the image sensor of the CCF camera to be used.

[Operation Timing] Next, the operation timing of the time-resolved fluorescence measurement performed using the apparatus shown in FIG. 1 will be described. First, pulse laser 1
The light emission timing, the delay timing by the delay circuit 12, and the image acquisition timing by the CCD camera 9 will be described with reference to a timing chart shown in FIG. As shown in FIG. 4, the laser light from the pulse laser 1 is irradiated during the light emission time t (the upper chart in FIG. 4). The delay circuit 12 generates a delay time τd in synchronization with the point 27 at which the emission of the pulse laser ends (the middle chart in FIG. 4). Further, the timing generator 13 generates an integration time τi in synchronization with the point 28 after the delay time τd (lower chart in FIG. 4). Only during this integration time τi,
The CCD camera 9 acquires a target image.

At this time, the light emission time t, the delay time τ
d and the integration time τi satisfy the following conditions. That is, t <τd <τi. Generally, the emission time t nanoseconds of the pulse laser is 1 to 5 nanoseconds (nsec), while the fluorescence lifetime of the delayed fluorescent substance is 1 μs to 500 μs (μsec). Therefore, the delay time τd is 1 μsec to 100 μsec, and the integration time is in the range of 10 μsec to 500 μsec. Also,
Each interval shown in FIG. 4, that is, the emission interval of the pulse laser (that is, the pulse width), the interval of two delay times, and
Have the following relationship: tl>τdl> τil. When acquiring a fluorescent image using a CCD camera as in the preferred embodiment of the present invention as described above, tl is set to 30 Hz in consideration of the video rate limit.
It is desirable that: However, the present invention is not limited to this, and each condition can be changed according to the properties of the delayed fluorescent substance used, the sample to be measured, and the like.

[Image Acquisition] Next, referring to FIGS.
Image acquisition will be described. The CCD camera 9 acquires an image only during the integration time τi. At this time, the fluorescent light generated from the sample passes through the objective lens 23, and passes through the desired filter 22, the imaging lens 21, and the intermediate variable power lens 20.
And reaches the CCD camera 9 (FIG. 3). The CCD camera 9 integrates the acquired fluorescence, and the integrated data is transmitted to the calculation / display unit 14 (FIG. 1), and is displayed on the display unit as a result.

[Delayed Fluorescent Substance] As the delayed fluorescent substance which can be used in combination with the apparatus of the present invention, any commonly used delayed fluorescent substance can be used.
For example, a substance having a functional group that reacts with an amino group or a phosphate group and a functional group that forms a complex with a lanthanoid ion in a molecule is included.

[Application] Although the preferred embodiments of the present invention have been described, the present invention is not limited to these examples, and various modifications can be made. For example, in the above-described example, an example has been described in which data is obtained as an image by integrating and imaging fluorescence obtained from a sample using a CCD camera, but instead of obtaining data as an image, a sample is scanned. This makes it possible to obtain the fluorescence intensity. Further, the obtained fluorescence intensity may be converted into a voltage signal by a photoelectric conversion unit such as a photomultiplier. It is also possible to set appropriate conditions for scanning by changing the frequency of the pulse laser and the pulse width.

[0030]

By using the time-resolved fluorescence measuring apparatus of the present invention, it is possible to minimize speckle noise caused by coherency, thereby obtaining a delayed fluorescence image without scattering noise. It is possible.
As a result, the present apparatus can provide highly sensitive and accurate fluorescence analysis means. In particular, for the first time with the provision of the device according to the invention, it is
It can be used for A chips and DNA microarrays. As a result, it becomes possible to selectively measure the target fluorescence intensity, and D
It has become possible to analyze NA chips and DNA microarrays with high sensitivity and high accuracy. That is, to minimize the effect of autofluorescence in the measurement system containing cells,
It becomes possible to selectively detect the fluorescence from the target DNA. It is also possible to minimize artifacts in the test.

[Brief description of the drawings]

FIG. 1 is a diagram showing a preferred example of a time-resolved fluorescence measuring device of the present invention.

FIG. 2 is a diagram showing a preferred example of an optical system used in the apparatus of the present invention.

FIG. 3 is a view showing a preferred example of an optical fiber bundle used in the apparatus of the present invention.

FIG. 4 is a diagram showing a chart showing pulse laser emission, delay, and timing of image acquisition.

[Explanation of Codes] Pulse laser 2. 2. Collimator lens 3. Optical fiber for light guide Optical fiber bundle 5. Optical fiber for light guide 6. Relay lens 7. 7. Reagent observation lamp Light source switching unit 9. CCD camera 10. Optical unit 1
1. Sample 12. Delay circuit 13. Timing generator 14. Calculation / display section 17. Light source switching mirror 18. Dichroic mirror 19. Relay lens 20. Intermediate zoom lens 21. Imaging lens 22. Filter 23. Objective lens 24. Convergent unit 25. Connection part 26. Bundle 27. End point of pulsed laser emission 28. Delay time end point

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Claims (3)

[Claims] 1. A time-resolved fluorescence detection device, comprising: an excitation light intermittent emission unit that emits an excitation light beam intermittently at a predetermined timing; an optical path for an excitation light beam emitted from the excitation light intermittent emission unit; A phase canceling unit provided on the optical path for canceling the phase of the excitation light beam, an optical system for acquiring fluorescence generated by excitation by the excitation light beam, and the excitation light intermittent emission unit and the optical system A time-resolved fluorescent light, comprising: a control unit configured to control an emission timing and an irradiation time of an excitation light beam by the excitation light intermittent emission unit, and a fluorescence acquisition timing by the optical system. Detection device. 2. The time-resolved fluorescence detection device according to claim 1, wherein the optical system measures the fluorescence as a fluorescence intensity image,
A time-resolved fluorescence detection device comprising a photoelectric conversion unit that converts the electric signal into an electric signal. 3. The time-resolved fluorescence detection device according to claim 1, wherein the phase eliminating unit is an optical fiber bundle.